Browsing by Issue Date, starting with "2020-01-08"
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- Gellan microspheres application for capture or purification of plasmid DNA vaccinePublication . Gomes, Diana Vanessa Duarte; Sousa, Ângela Maria Almeida de; Passarinha, Luís António PaulinoCervical cancer is the 4th cause of death among women worldwide and is profoundly associated with HPV infection, due to apoptosis inhibition and uncontrolled cell proliferation caused by oncoproteins E6 and E7 action. At the moment, some prophylactic vaccines are available in the market, but that are only capable of preventing HPV infection. Thus, the development of effective treatment for HPV-infected individuals is fundamental. DNA vaccines emerged as a promising way to prevent and treat several diseases since it can stimulate both cellular and humoral immune responses. The biotechnological process for obtaining plasmid DNA (pDNA) includes several steps, which present an environmental impact and makes it quite expensive to the pharmaceutical industry. Therefore, it is crucial to explore new alternatives. In this work, copper-crosslinked gellan microspheres were produced through a water-in-oil emulsion in order to capture pDNA directly from the Escherichia coli (E. coli) lysate seeking a reduction in the recovery and clarification-associated costs. The lowest diameter of gellan microspheres was achieved with 1.41 % of an aqueous gellan gum solution, previously heated at 90ºC, and dripped through a syringe to the oil solution formerly heated at 100 ºC with constant stirring of 750 rpm at a flow rate of 75 µL/min. Afterwards batch method optimization, the gellan microspheres captured 15.61 % of pDNA with 2.42 % of purity by a strategy based on immobilized metal affinity chromatography (IMAC), by manipulating the pH and ionic strength of binding and elution buffers. Another strategy was developed in order to increase the pDNA capture by precipitating the E. coli lysate with ammonium sulfate. The elimination of major impurities improved the recovery percentage to 32.41 % and the purity degree to 12.43 %. Moreover, copper-crosslinked gellan microspheres were functionalized with polyethylenimine (PEI), in order to increase the pDNA capture by increasing the functional groups in the microspheres surface. This allowed an improvement in the recovery percentage to 88.09 %, but the same did not happen to the purity percentage, 3.18 %. Thus, if the central aim is total pDNA capture from crude lysates without resorting to salts or organic solvents, the strategy in which the microspheres were functionalized with PEI showed great potential. On the other hand, if the main objective is to capture pDNA with higher purity, it is recommended to perform a prior step to the capture with ammonium sulfate, where copper-crosslinked microspheres may be applied or also the ones functionalized with PEI. In conclusion, these simple, fast and low-cost strategies allow lysate clarification since an E. coli lysate usually has 1.07 % of pDNA.
- Aircraft Attitude Tracking using a Model Reference Adaptive ControlPublication . Castanho, Emanuel da Costa; Bousson, KouamanaA discrete-time explicit Model Reference Adaptive Control (MRAC) with constant trace algorithm is applied to a linearized aircraft model during longitudinal and lateral-directional motions in order to test the performance of this type of control during specific flight conditions. The model was obtained through system identification with data generated from the linearized state equations of the F-4C. In the longitudinal case the aircraft behaves like a Single-Input-Single-Output (SISO) system and simulations are performed for two examples of pitch angle data, in which two expressions for the control (classic and penalized) are applied in each example to compare their performance. In the lateral-directional case the airplane behaves like a Multi-Input-Multi-Output (MIMO) system with equal number of inputs and outputs and the MRAC control law must be modified to describe a decoupling process. Simulations are performed in order to verify if the controller is able to handle the coupling relation between some variables, such as lateral velocity, roll angle, aileron angle and rudder angle. The adaptive control in both study cases and for the chosen initial conditions showed good tracking when following the reference output, presenting no drift problems. The choice of the initial simulation conditions is also analyzed, in order to prevent actuator saturation.
- Proteomic study of the therapeutic effect of a DNA vector on cervical cancer cellsPublication . Pinto, Adriana Resende; Sousa, Ângela Maria Almeida de; Passarinha, Luís António PaulinoCancer is a major global health problem, accounting for more than 8 million deaths/year worldwide. In particular, cervical cancer is the 3rd most common malignancy and 4th cause of death among women globally, being a persistent High Risk-HPV infection the main factor for cervical cancer development. E6 and E7 HPV oncoproteins are responsible for cancer development by degrading and inhibiting p53 and pRb tumour suppressor proteins, respectively. Moreover, microRNAs (miRs) have been found to regulate tumorigenesis. In fact, miR-375 has the ability to silence HPV E6 and E7 oncoproteins. Gene therapy is a promising strategy to treat acquired diseases and/or genetic disorders, aiming to deliver genetic material into target cells or tissue, expressing it to induce a therapeutic effect. Minicircle DNA (mcDNA) is a new biopharmaceutical product only composed by the eukaryotic transcription unit, improving its safety and therapeutic effect, obtained through intramolecular recombination of the parental plasmid. Thus, this work aims to produce and purify a mcDNA vector encoding primiR-375 and p53 genes to silence E6 and E7 oncoproteins and re-establish p53 and pRb tumour suppressor levels on cervical cancer cells. The purification of mcDNA vectors was performed using size exclusion chromatography sepharose columns (Sephacryl S-1000 SF), showing that for the smallest vector, mcDNAprimiR-375, a 106 mL column allowed the efficient recovery of chromatographic fractions composed only with mcDNA. On the other hand, for the biggest mcDNA vectors, it was necessary to exploit the parameters affecting the sample molecules separation, choosing to use a column with 180 mL of volume, allowing, similarly to the mcDNA-primiR-375 purification, recover fraction only composed with mcDNA. Simultaneously, cytotoxicity assays on human fibroblasts and CaSki cells were performed, confirming that none of the vectors were toxic to the fibroblasts and the mcDNA primiR-375+p53 presented the lower cell viability for CaSki cells. In addition, the proliferation assay results performed on CaSki cells show that number of viable cells decreases for the mcDNA vectors transfected cells, corroborating the cytotoxicity assays results for this cell line. Western-Blot confirmed the re-establishment of tumour suppressor proteins levels after 48h of transfection using mcDNA-p53 and mcDNAprimiR-375+p53. Overall, these results suggest that the use of DNA vectors encoding for more than one therapeutic gene, for example, the mcDNA-primiR-375+p53, allowed to achieve, on in vitro assays, results suggesting a possible faster therapeutic action, thus demonstrating that they may revolutionize their application in targeted therapies.
- Tingimento de fibras têxteis com prodigiosina produzida por Serratia plymuthicaPublication . Costa, Ana Rita de Sousa Vieira da; Gouveia, Isabel Cristina Aguiar de Sousa e SilvaNos tempos de hoje existe uma maior consciencialização no que diz respeito à fragilidade ambiental e respetivas fontes prejudiciais. A área têxtil é uma das indústrias mais poluidoras a nível mundial. Com este facto em foco, surgiu este trabalho em que se aliou a área têxtil à biotecnologia, para produzir um processo de tingimento mais ecológico e sustentável, simultaneamente eficiente que permita satisfazer as necessidades impostas pela população mundial. Com a preocupação ecológica surgiu uma maior apreensão com os corantes sintéticos, considerados tóxicos tanto para o Homem como para o ambiente, e voltou a emergir a investigação na área dos pigmentos naturais. Estes podem ser de origem vegetal, mineral, animal e microbial, e tendo em conta o balanceamento de vantagens e desvantagens de todas as classes, os pigmentos microbianos ganham a preferência, principalmente os bacterianos. A realização do presente estudo serviu para investigar a capacidade tintorial do extrato bruto de prodigiosina, produzido por S.plymuthica, de uma forma ecológica. Avaliou-se a influência de diferentes parâmetros experimentais (temperatura de tingimento; adição de auxiliares; adição de mordentes e pH) e também a interação do pigmento com diferentes fibras (acetato, algodão, poliamida, poliéster, acrílica e lã), com base na leitura espetrofotométrica das cores obtidas, pelos valores de intensidade colorística (K/S) e pelos valores de igualização (dE) correspondentes. Em fases posteriores, na produção de um tingimento otimizado, caracterizaram-se as cores obtidas nas diferentes fibras, através das coordenadas CIELab; avaliaram-se as propriedades de solidez com base na lavagem doméstica e industrial, na fricção a seco e a húmido e à luz; determinou-se a resistência à abrasão segundo o método de Martindale; avaliou-se, ainda, a tensão e o alongamento de rutura e finalmente a atividade antibacteriana, em virtude das conhecidas propriedades bioativas do pigmento utilizado. A otimização dos parâmetros tintoriais resultou nas condições ideais para poliamida, com a temperatura de tingimento a 60ºC, mordentagem com um bio-mordente como a L-Cysteína (LCys) a 3% e com a solubilização parcial da prodigiosina em etanol. O uso deste extrato bruto de pigmento bacteriano exibiu resultados muito positivos e vantajosos em relação aos corantes sintéticos e à prodigiosina extremamente purificada, resultando numa estratégia potencialmente mais ecológica para o tingimento destas fibras.